Cholangiocyte proliferation/loss is a typical hallmark of cholestatic liver diseases specifically targeting different sized cholangiocytes. Our studies are a direct outgrowth of our continuing efforts to understand the intracellular mechanisms regulating the functional heterogeneous responses of small and large intrahepatic cholangiocytes to gastrointestinal hormones and liver injury/toxins. While the function of large cholangiocytes is regulated by activation of cAMP-dependent signaling, the pathophysiology of small cholangiocytes (which has been postulated to be regulated by the IP3/Ca2? I-dependent signaling) is undefined. Secretin receptor (SR) has been suggested to play a role in the regulation of cholangiocyte growth/loss since there is: (i) functional increased expression of SR parallel to enhanced cholangiocyte hyperplasia; and (ii) decreased SR expression and secretin-stimulated cholangiocyte secretion in pathological states associated with damage of bile ducts. However, direct evidence for the role of secretin and its receptor (expressed only by large cholangiocytes in normal rodent liver) in the regulation of cholangiocyte heterogeneous growth/loss is lacking. We propose the key hypotheses that: (i) secretin is a trophic factor (secreted by cholangiocytes) that activates the growth of normal and cholestatic (during BDL) large cholangiocytes (the only hepatic cell type expressing SR) by an autocrine mechanism via activation of cAMP-dependent signaling; (ii) secretin is an autocrine protective factor against CCl4-induced damage of large cholangiocytes; (iii) in vivo (in KO mouse models) and in vitro (in small and large cholangiocytes) silencing of the secretin gene and its receptor reduces large cholangiocyte growth (e.g., in response to BDL), and exacerbates the damage of large ducts in response to CCl4; and (iv) small cholangiocytes proliferate and secrete by both [a.] the upregulation of IP3/Ca2? I signaling (that is constitutively expressed by normal small cholangiocytes), and [b.] the de novo acquisition of large cholangiocyte phenotypes such as the expression and synthesis of secretin (through activation of NeuroD1 and SP1), and secretin receptor (by activation of CaMK I and the adenylyl cyclase, AC8, and the subsequent activation of CREB and SP1/3). The proposed studies suggest that the coordinated expression of Ca2+ and cAMP-dependent phenotypes (by small cholangiocytes) may be important to replenish the biliary tree during damage of large ducts by liver injury/toxins. To test this hypothesis, we have designed three specific aims to: (i) demonstrate that secretin is a trophic factor for cholangiocytes, and that secretin differentially regulates the growth/loss of small and large cholangiocytes by an autocrine mechanism in normal and pathological conditions; (ii) define that in vivo and in vitro molecular manipulation of the secretin receptor gene ablates the proliferative and apoptotic responses of small and large cholangiocytes to cholestasis and liver injury; and (iii) To define the in vitro intracellular mechanisms regulating secretin and secretin receptor expression during the proliferative/apoptotic response of small and large cholangiocytes to cholestasis and liver injury. We will use a number of in vivo (secretin and SR KO mouse models), in situ (e.g., immunohistochemistry in liver sections), and in vitro molecular (e.g., silencing, and real-time PCR) and cellular (isolated and cultured small and large murine cholangiocytes) tools in conjunction with biochemical and immunological approaches to pinpoint the intracellular mechanisms by small and large cholangiocytes differentially proliferate or are lost in response to liver injury/damage. The proposed studies will introduce the novel concept that cholangiocytes secrete the hormone secretin, and that manipulation of secretin levels in cholangiocytes may be important in the management of the balance between cholangiocyte growth/loss in cholangiopathies. PUBLIC HEALTH RELEVANCE: Management of liver diseases represents one of the major challenges of the Veterans Administration. There is a high risk and incidence of cholestatic liver diseases due to alcohol and hepatitis viruses in Veterans, which is one of the most common reasons for hospitalization and mortality in American Veterans. Damage of bile ducts is a pathological process that is observed in virtually all cholangiopathies (such as, drug induced ductopenia, primary biliary cirrhosis and primary sclerosing cholangitis). Understanding the mechanisms by which bile ducts proliferate in normal and diseased states (which the proposed studies directly address) will likely lead to new therapeutic approaches and a reduction of morbidity and mortality in American Veterans with liver diseases.